BACKGROUND: To investigate the epidemiological and phenotypic characteristics and molecular relatedness of L. monocytogenes, which were cultured from the blood and cerebrospinal fluid (CSF) samples isolated from two neonates. METHODS: In the present case study, two infected neonates were interviewed and epidemiological investigation performed. The phenotypic characteristics and molecular relatedness of L. monocytogenes was characterized by serotyping, pulsed-field gel electrophoresis and whole-genome sequencing (WGS). RESULTS: The field investigation found that the two neonates were born in the same hospital (Hospital B) and admitted to the neonatal department through different channels within half an hour by different nurses, where they were weighed and placed in different but adjacent incubators. Then they were cared for by the same group of nurses that evening. It is worth noting that there was no record of sanitation of the neonatal incubator of neonate-1. The serotype of the two isolated L. monocytogenes were 1/2b, with an indistinguishable pulsotypes and were sequence type (ST) 87. WGS showed that there were no core SNP differences identified. In order to explore the genomic traits associated with L. monocytogenes virulence genes, we identified the Listeria pathogenicity island 4 and found that the genome was devoid of any stress islands. There are no positive results from the environmental samples. Considering the genomic data together with epidemiological evidence and clinical symptoms, insufficient surface cleaning along with the nursing staff caring for these neonates was considered as cross-infection factors. CONCLUSIONS: To our knowledge, this is the first report of a nosocomial cross-infection of L. monocytogenes ST87 between two neonates, which carries the recently identified gene cluster expressing the cellobiose-family phosphotransferase system (PTS-LIPI-4) between two neonates. The test results of environmental samples in the hospital indicate that strict sterilization and patient isolation measures cannot be emphasized enough in neonatal nursing.